Chromatin insulators are DNA-protein complexes situated throughout the genome that are proposed to contribute to higher order organization and demarcation into distinct transcriptional domains. Mounting evidence in different species implicates RNA and RNA-binding proteins as regulators of chromatin insulator activities. Here we identify the Drosophila hnRNP M homolog Rumpelstiltskin (Rump) as an antagonist of gypsy chromatin insulator enhancer-blocking and barrier activities. Despite ubiquitous expression of Rump, decreasing Rump levels leads to improvement of barrier activity only in tissues outside of the central nervous system (CNS). Furthermore, rump mutants restore insulator body localization in an insulator mutant background only in non-CNS tissues. Rump associates physically with core gypsy insulator proteins, and ChIP-Seq analysis of Rump demonstrates extensive colocalization with a subset of insulator sites across the genome. The genome-wide binding profile and tissue-specificity of Rump contrast with that of Shep, a recently identified RNA-binding protein that antagonizes gypsy insulator activity primarily in the CNS. Our findings indicate parallel roles for RNA-binding proteins in mediating tissue-specific regulation of chromatin insulator activity. Here we introduce metaseq, a software library written in Python for manipulating and visualizing data from high-throughput sequencing studies. We demonstrate its use by analyzing existing and new data sets related to chromatin insulators. Recent studies in Drosophila and mammals have implicated associated RNAs in regulation of chromatin insulator activities. The Drosophila RNA-binding protein Shep has been shown to antagonize gypsy insulator activity in a tissue-specific manner, but the precise role for associated RNA in this process remains unclear. In this study, we use metaseq to analyze and compare RNA immunoprecipitation and sequencing (RIP-seq) data for Shep and the core gypsy insulator protein Su(Hw) with chromatin occupancy (ChIP-seq) data for both factors in two different cell types, and integrate these data with publicly available ChIP-chip and RNA-seq data. Based on the metaseq-enabled analysis presented here, we propose a model where Shep binds in cis to associated transcripts, then is recruited to insulator complexes in trans where it plays a negative role in insulator activity.